Imaging, Diagnosis, PrognosisSee commentary p. 203

Plectin-1 as a Novel Biomarker for Pancreatic Cancer

Dirk Bausch1, Stephanie Thomas2, Mari Mino-Kenudson3, Carlos Fern�andez-del Castillo1,Todd W. Bauer4, Mark Williams2,5, Andrew L. Warshaw1, Sarah P. Thayer1, and Kimberly A. Kelly2

AbstractPurpose: We are in great need of specific biomarkers to detect pancreatic ductal adenocarcinoma (PDAC)

at an early stage, ideally before invasion. Plectin-1 (Plec1) was recently identified as one such biomarker.

However, its suitability as a specific biomarker for human pancreatic cancer, and its usability as an imaging

target, remain to be assessed.

Experimental Design: Specimens of human PDAC, chronic pancreatitis, and normal pancreata were

evaluated by immunohistochemistry and Western blot analysis. To validate Plec1 as an imaging target,

Plec1-targeting peptides (tPTP) were used as a contrast agent for single photon emission computed

tomography in an orthotopic and liver metastasis murine model of PDAC.

Results: Plec1 expression was noted to be positive in all PDACs but negative in benign tissues. Plec1

expression increases during pancreatic carcinogenesis. It was found to bemisexpressed in only 0% to 3.85%

of early PDAC precursor lesions (PanIN I/II) but in 60% of PanIN III lesions. Plec1 expression was further

noted to be retained in all metastatic foci assayed and clearly highlighted thesemetastatic deposits in lymph

nodes, liver, and peritoneum. In vivo imaging using tPTP specifically highlighted the primary andmetastatic

tumors. Biodistribution studies performed after imaging show that the primary pancreatic tumors and liver

metastases retained 1.9- to 2.9-fold of tPTP over normal pancreas and 1.7-fold over normal liver.

Conclusions: Plec1 is the first biomarker to identify primary and metastatic PDAC by imaging and may

also detect preinvasive PanIN III lesions. Strategies designed to image Plec1 could therefore improve

detection and staging. Clin Cancer Res; 17(2); 302–9. �2010 AACR.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourthleading cause of cancer-related deaths in the United Statesand other industrialized countries (1). Despite manyefforts, it remains a devastating disease with a 5-yearsurvival rate of less than 5% and a median survival of lessthan 1 year (1). This grim prognosis is mostly due to thecancer’s aggressive biological behavior with early invasionand metastasis, leading to an initial diagnosis at anadvanced incurable stage in more than 80% of patients(2). The only potentially curative treatment is radical

surgical resection (3). Prognosis after resection dependson tumor size (<3 cm), lymph node involvement, andstatus of the resection margin (3).

Early diagnosis of small or even preinvasive cancersbefore the onset of metastasis is currently the only meansto substantially improve resectability, prognosis after resec-tion, and ultimately survival (4). However, all availablediagnostic tools and biomarkers for PDAC fail to detectearly or preinvasive cancer and suffer from low specificityand sensitivity (5, 6). The only clinically available serumbiomarker for PDAC is CA 19-9, which is of limited use (7).Invasive endoscopic procedures (endoscopic ultrasoundand endoscopic retrograde cholangiopancreatography)suffer from potential for injury to the pancreas and arehighly operator dependent (5, 6, 8, 9). Cross-sectionalabdominal imaging is not reliable enough to allow forscreening of high-risk patient populations (10, 11) andfails to detect metastases in up to 30% of patients pre-operatively (12, 13). All 3 screening modalities also cannotsafely discriminate PDAC from chronic pancreatitis (CP),which is of particular importance because PDAC can arisein the background of CP, and CP often mimics PDAC dueto their similar clinical signs and symptoms (14, 15). Thissimilarity makes the identification of biomarkers that dis-tinguish PDAC from CP very challenging (16).

PDAC is believed to progress through precursor lesionstermed pancreatic intraepithelial neoplasia (PanIN).

Authors’ Affiliations: 1Department of Surgery, Massachusetts GeneralHospital and Harvard Medical School, Boston, Massachusetts; 2Depart-ment of Biomedical Engineering, University of Virginia, Charlottesville,Virginia; 3Department of Pathology, Massachusetts General Hospitaland Harvard Medical School, Boston, Massachusetts; and Departmentsof 4Surgery and 5Radiology, University of Virginia, Charlottesville, Virginia

Corresponding Authors: Kimberly Kelly, Department of Biomedical Engi-neering, University of Virginia, Box 800759 Health System, Charlottesville,VA 22908. Phone: 434-243-9352; Fax: 434-982-3870; E-mail: kak3x@virginia.edu or Sarah P. Thayer, W. Gerald Austen Scholar in AcademicSurgery, Department of Surgery, Massachusetts General Hospital andHarvard Medical School, 15 Parkman Street, WACC 460, Boston, MA02114-2622. Phone: 617-726-0624; Fax: 617-726-0630.E-mail: Sthayer@partners.org

doi: 10.1158/1078-0432.CCR-10-0999

�2010 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(2) January 15, 2011302

Early PanIN I and II lesions are frequently observed innormal pancreata and CP. PanIN III lesions are consid-ered carcinoma in situ (preinvasive cancer) and possessmany of the genetic aberrations of invasive cancer. Thedetection of PanIN III lesions has a direct impact onclinical treatment decisions (17–19). Thus, the idealbiomarker for PDAC should not only differentiate benignconditions (CP) from malignancy, but also be able todetect small cancers, ideally at the preinvasive PanIN IIIphase. Novel biomarkers that satisfy these demands havebeen difficult to identify, and none of the potentialcandidate biomarkers discovered so far have been ableto meet all of the above criteria (20–24). On the basis offindings in vitro and in a genetically engineered mousemodel, Plectin-1 (Plec1) was recently suggested as abiomarker for PDAC (25), but its suitability as a biomar-ker for primary and metastatic human PDAC and itsprecursor lesions, and its capability to differentiate PDACfrom CP, remain to be assessed.This study shows that Plec1 is not only a biomarker for

human invasive and metastatic PDAC, but may also serveas a marker for preinvasive PanIN III lesions. Plec1 alsodistinguishes malignant pancreatic disease from CP. Inpreclinical orthotopic mouse models of PDAC, Plec1overexpression can be exploited for noninvasive imagingof PDAC and its metastases. These data suggest that Plec1may indeed be an ideal biomarker for small and prein-vasive cancers and that Plec1-targeted imaging of PDAC isfeasible. Clinical use of Plec1-based imaging should per-mit the early diagnosis of small or even preinvasivecancers in addition to metastases, potentially leading toimproved resectability rates and survival.

Materials and Methods

Tissue samplesAll tissues and biological samples were collected with

the approval and in accordance with the requirements ofthe Institutional Review Board of the Massachusetts Gen-eral Hospital, Boston, Massachusetts.

Paraffin-embedded tissue samples were obtained fromthe files of the Department of Pathology of the Massachu-setts General Hospital, Boston, Massachusetts. All speci-mens had an established diagnosis at the time ofassessment. A total of 4 normal pancreata, 15 CP, 14 PanINI, 26 PanIN II, 15 PanIN III, 41 PDAC, 8 liver metastasis, 11lymph node metastasis, 10 with matching primary tumors,and 9 peritoneal metastasis were obtained. For the assess-ment of Plec1 expression in extrapancreatic human cancer,a commercial tumor tissue microarray (MTU951, US Bio-max) was used.

Mice and cell linesAll animal procedures were approved by the University

of Virginia Animal Care and Use Committee and theMassachusetts General Hospital Subcommittee onResearch Animal Care. Nude mice (nu/nu) were purchasedfrom the National Cancer Institute. FVB/NJ mice werepurchased from the Jackson Laboratory. Mice were main-tained in a germ-free environment and had access to foodand water available ad libitum.

The L3.6pl pancreatic cancer cell line was originallyderived from a repeated cycle of injecting COLO-357 cellsinto the pancreas of nude mice, selecting for liver metas-tases, and reinjecting into the pancreas. AK134 cells werederived from spontaneous PDAC arising in Pft1-Cre; LSL-K-RasG12D; p53þ/�mice in the background of an inbredFVB/NJ strain. The Panc1 pancreatic cancer cell line wasobtained from ATCC. All cell lines were routinely verifiedby morphology and growth curve analysis, and tested forMycoplasma.

Animal modelsThree orthotopic mouse models and 1 mouse model of

liver metastasis were employed to assess the suitability ofPlec1 as an in vivo imaging biomarker. L3.6pl (1� 106, n¼10) or Panc1 cells (11�106,n¼5) in 50mLHank’s BufferedSterile Saline (HBSS) were injected into the head of thepancreas of nu/nu mice. AK134 (2.5 � 105) were injectedinto the pancreas of FVB/NJ mice (n ¼ 8). To obtain livermetastasis, 2.5�105 AK134 cells (n ¼ 5) in 50 mL of HBSSwere injected into the capsule of the spleen. Seven days(AK134), 10 days (L3.6pl), 4 weeks (Panc1), or 15 days(AK134 liver metastasis) after injection, animals wereimaged, sacrificed, and tetrameric synthetic peptide (tPTP)biodistribution assessed. All animals underwent grossinspection of the abdominal cavity and liver for metastasis.Histology was used to confirm macroscopic findings. Ascontrol animals, FVB/NJ (n¼ 2) or nu/nu (n¼ 5)micewereinjected with 50 mL of HBSS into the pancreas and spleenand then imaged 1 to 4 weeks after injection.

Clinical Relevance

Specific biomarkers for the detection of pancreaticductal adenocarcinoma (PDAC) at an early or preinva-sive stage are currently unavailable. Here we report on apancreatic cancer biomarker, Plectin-1, that distin-guishes PDAC from benign inflammatory diseases suchas chronic pancreatitis. Plectin-1 is identified in 100%oftested PDAC tumors and 60% of preinvasive PanIN IIIlesions, and is retained in metastatic deposits, charac-teristics needed for an ideal imaging biomarker. In vivoimaging in orthotopic and liver metastases models ofpancreatic cancer using a Plec1-targeted imaging agentfor single photon emission/CT resulted in enhanceddetection not only of the primary tumor but also ofsmall peritoneal and liver metastases. These data suggestthat Plectin-1 is a specific novel imaging biomarker forPDAC. Although further translational studies inhumans will be needed, this study shows that it canbe incorporated into present day imaging technologiesused in humans and that this target may be used forimproved early detection and staging.

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Western blot analysisPancreatic tissue (50mg) obtained as snap-frozen surgical

specimens was homogenized in RIPA buffer [50 mmol/LTrizma Base (pH 7.4), 1% Triton X-100, 0.25% sodiumdesoxycholate, 100 mmol/L EDTA, 150 mmol/L NaCl] incombination with a protease inhibitor cocktail (0.001 mg/mL aprotinin, bestatin, pepstatin, leupeptin, and 0.005 mg/mL20mmol/L PMSF; Sigma-Aldrich). The lysate was clearedby centrifugation. To ensure equal loading, a highly sensitiveand precise copper-based assay was used to determine theprotein concentration of each sample (2-DQuant Kit; Amer-sham Biosciences). A portion of 20 mg of protein per lanewere separated via SDS-PAGE and transferred onto a nitro-cellulose membrane. Equal transfer was verified by Ponceaustaining. Antigen detection was done using a rabbit mono-clonal antibody against human Plec1 (Abcam). The second-ary antibody was a HRP-coupled goat anti-rabbit polyclonalantibody (Sigma-Aldrich). Bands were visualized withenhanced chemoluminescence (control: rat brain lysate;Santa Cruz Biotechnology).

Immunohistochemistry for Plectin-1Paraffin-embedded sections were deparaffinized,

hydrated with Tris-buffered saline, and blocked withH2O2. Antigen retrieval was achieved by boiling tissue inRetrievit (BioGenex). After blocking with avidin/biotin(http://www.vectorlabs.com/contactus.asp#contact VectorLaboratories) and 5% goat serum in Tris-buffered saline,slides were incubated overnight at 4�C with 1:250 Plec1antibody (Abcam). Sections were washed 3 times inTris-buffered saline with Tween, followed by incubationwith biotinylated anti-rabbit goat secondary antibody(http://www.vectorlabs.com/contactus.asp#contact VectorLaboratories), than developed using DAB (Invitrogen) andcounterstained with hematoxylin. Slides were evaluatedusing a Y-FL microscope (Nikon).

Expression of Plec1 in nerves within each slide wasused as a staining control and reference for stainingintensity. Nerves were noted to have a moderate stainingintensity. Staining intensity was recorded by 2 indepen-dent observers, and in case of discrepant results, evalu-ated by a third observer. Plec1 staining was classified asnegative if the staining intensity was weaker than nerves.It was classified as positive if the staining was as least asstrong as nerves.

In vitro competition assayTetrameric Plec1-targeted peptide [tPTP-4(bAKTLLPTP-

GGS(PEG5000))KKKDOTAbA-NH2)] was synthesized in aGMP grade facility (CS Bio Company). As a control, non-binding tetramer [ncPTP-4(bAKHVMSKQGGS(PEG5000))KKKDOTAbA-NH2)] was also synthesized. For Indium label-ing,peptide(100mg)wasdissolved in20mLPBS, thendilutedin 100 mL ammonium acetate buffer (0.1 mol/L, pH 4.5).Indiumchloride(5mCi inwater;CardinalHealth)wasmixedwith the peptide and allowed to equilibrate with mixingat 40�C for 15 minutes. The reaction mixture was purifiedby size exclusion using a PD10 desalting column pre-

equilibrated with Dulbecco’s phosphate-buffered saline.For in vitro peptide validation experiments, cells were incu-batedatroomtemperature for1hourwithtPTPorncPTPwithconcentrations ranging from 10�3 to 10�9 and 5 mCi tPTP-In111 intriplicate.After1hour, thecellswerewashedandlysedwith 100 mL 1 mol/L NaOH for 5 minutes. The mixture wasthen transferred to tubes and activity analyzed on a gammacounter.

ImagingMicewere injectedwith1mCiof 111In labeled tPTP, then

imaged 4 hours injection with a microSPECT/CT scannerdesigned and built at UVa. Computed tomography (CT)acquisition used 200 evenly spaced projections spanning200 degrees over approximately 5 minutes. Pinhole singlephoton emission computed tomography (SPECT) scanningwas then performed using 2 opposing gamma camerassimultaneously. The 2 cameras were fitted with 0.5 mmdiameter tungsten pinholes. Sixty evenly spaced projectionviews per camerawere obtained over 180 degrees, for a totalof 120 views at 3-degree increments over 360 degrees. TheSPECT acquisition timewas approximately 45minutes. Thereconstructed CT voxel size was 0.082� 0.082� 0.082mmon a 640 � 640 � 768 image matrix. The reconstructedSPECT voxel size was 0.65 � 0.65 � 0.65 mm on an 80 �80 � 80 image matrix. All SPECT images were corrected forradioactivity decay but not for gamma ray attenuation.

Biodistribution and blood half-lifeAfter mice were imaged via SPECT/CT, animals were

sacrificed and their organs harvested and placed into pre-weighed Eppendorf tubes. Each tube was then reweighed todetermine the weight of the organ and the radiation mea-sured. Tubes containing organs were analyzed on a gammacounter. To determine the plasma lifetime of the probe, amouse injected with the tPTP-Peg-111In was bled 0, 15, 30,45, 60, and 120 minutes postinjection, and the sampleanalyzed on a gamma counter. Tissue samples were thenplaced in histology cassettes and fixed for paraffin embed-ding. After the radioactivity in the tissue samples decayed,the blocks were sectioned on amicrotome and evaluated byhematoxylin and eosin (H&E).

Results

Plec1 expression intensity and pattern distinguishmalignant from benign pancreatic disease

To determine whether Plec1 can be used as a markerfor the detection of PDAC, immunohistochemistry (IHC)of human tissue sections was performed. Plec1 expressionwas scored as negative in all benign tissues (4 of 4 normalpancreata and 15 of 15 CP). In contrast, 41 of 41 PDACstained strongly positive for Plec1 (Fig. 1 A and B). Simi-larly, Western blotting of pancreatic tissue lysates detectedno Plec1 in normal pancreas or CP whereas it is presentin each lysate from PDAC (Fig. 1C). Thus, Plec1 is identi-fied in all PDACs and clearly distinguishes malignant frombenign pancreatic disease (Fig. 1).

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A

B

C

Figure 1.Plec1 immunohistochemistry andWestern blot. A, representative images of the evaluated normal pancreata, chronic pancreatitis (CP), PanIN, PDAC,xenografted PDAC, and PDAC metastasis sites (liver, lymph node, and peritoneum). Overview (top) and detailed view of the black box (bottom).Chronic pancreatitis and normal pancreas do not express Plec1. PanIN III has a membranous staining pattern. PDAC and PDAC xenograft tissue stainmoderately to strongly cytoplasmic andmembranous for Plec1. CommonPDACmetastasis sites do not show significant Plec1 expression, whereas the tumorcells stain intensely for Plec1. B, distribution of staining intensity and staining pattern in the specimens. All PDAC cases were Plec1-positive, whereas normalpancreas and CP did not express Plec1. All PanIN I and most II lesions were Plec1-negative, whereas the majority of PanIN III lesions were Plec1-positive.The cellular localization of Plec1 also changes during carcinogenesis. The protein is found only in the membrane in 33% of PanIN III lesions, whereas 27%of PanIN III and all PDAC show membranous and cytoplasmic PLec1 expression. C, quantitative Western blot for Plec1 from 50 mg of pancreatic tissue(snap-frozen surgical specimens). No Plec1 was detected in the normal pancreas and CP, whereas it was present in each PDAC.

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To determine whether Plec1 expression changes duringcarcinogenesis, tissue sections of PDAC precursor lesions,PanINs, were evaluated by IHC. Although 0% PanIN Iand only 3.85% PanIN II are Plec1-positive, 60% ofPanIN III and 100% of invasive PDAC (Fig. 1A and B)were Plec1-positive. Plec1 expression thus increases dur-ing pancreatic carcinogenesis and discriminates early-stage PanIN I and II lesions from PanIN III and PDAC.During carcinogenesis, the cellular localization of Plec1also changes. Plec1 was identified in the cytoplasm and/or on the cell membrane. Although its expression wasrestricted to the cell membrane in 33% of PanIN III (5/13), a membranous and cytoplasmic expression of Plec1was observed in 26.67% of PanIN III (4 of 13) and 100%invasive PDAC (41 of 41; Fig. 1C). Sensitivity and spe-cificity of Plec1 for differentiating PanIN III and PDACfrom normal pancreata, CP, and lower-grade PanINlesions were 87% and 98%, respectively. Sensitivity forinvasive cancers (PDAC) alone was 100%.

Plec1 is not expressed in most normal human tissueand is retained in PDAC metastasis

IHC of a human tissue microarray revealed that Plec1 isnot expressed by most normal tissue, with the exception ofthe skin and genitourinary tract (Fig. 2). Specifically, it isnot expressed in the liver, lymph node, lung, or perito-neum. PDAC has a propensity to metastasize early to thesesites. To evaluate the suitability of Plec1 as a biomarker formetastatic disease, IHC of metastatic deposits was per-formed. All metastatic foci assayed retained their Plec1expression, clearly identifying and highlighting metastatic

deposits in the liver (8 of 8), lymph nodes (11 of 11), andperitoneum (9 of 9). The 10 lymph node metastases hadthe same pattern and staining intensity as their matchedprimary tumor (Fig. 1A ).

Plec1 is an ideal biomarker for detecting pancreaticcancer, but may also be an ideal biomarker for detectingother cancers, such as esophageal, stomach, and lungcancers, where a differential expression between normaland cancerous tissue was also noted in the tissue micro-array. The discovery that Plec1 can be used to highlighttumors and their potential metastatic foci suggests thatsmart imaging agents targeting this marker could be usedto improve diagnosis and staging (Fig. 2).

Plec1-targeting probes can be used for noninvasiveimaging of PDAC

To determine whether Plec1 can be used as an imagingbiomarker to facilitate the detection of human PDACin vivo, we employed Plec1-targeted peptides derived froma phage display screen (25) to synthesize a tPTP thatfunctions as a clinically relevant imaging agent for SPECT.In vitro validation of the specificity of the tPTP wasperformed by competition assay with labeled tPTP andnonrelated control PTP (nrPTP). The Ki (inhibition dis-sociation constant) for tPTP was 8.3 � 10�7 mol/l versus2.86 � 10�6 mol/L for nrPTP (Fig. 3A). Animals bearingorthotopically injected human pancreatic cancer cells(L3.6pl or Panc1) were administered tPTP and imagedvia SPECT/CT 4 hours after tPTP injection. In both L3.6pland Panc1 animals, imaging illuminated the tumorin the pancreas (Fig. 3B and data not shown). Panc1

Figure 2. Plec1 expression in normal and malignant human tissue: Immunohistochemistry of a tissue microarray evaluated for Plec1 expression. Most normaltissue shows only weak Plec1 expression. A clear difference in Plec1 expression distinguishing normal from malignant disease is observed in the pancreas,esophagus, stomach, and lung. Common PDAC metastasis sites (lymph node, liver) do not express Plec1.

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orthotopically injected animals did not form metastaticdisease. Consistent with this finding, no tPTP uptakewas identified outside the pancreas. However, 2 of 10mice injected with L3.6pl at autopsy were found to haveperitoneal metastases. tPTP SPECT/CT imaging wasable to accurately detect the peritoneal metastases in these2 animals (Fig. 3B, L3.6pl). Likewise, in a syngeneicmouse model of PDAC, tPTP was able to highlight theprimary tumor and associated metastases in the perito-neum (Fig. 3B, AK134). In contrast, only the kidneys werevisible in control animals (Fig. 3B, null).To validate and quantitate imaging results, biodistribu-

tion studies were performed to confirm tumor-specifictPTP accumulation. Biodistribution results showed thatthe pancreatic tumors from all 3 cell lines had a statis-tically significant 1.9- to 2.9-fold higher uptake whencompared with pancreata from control animals(Fig. 3C, P < 0.01). The probe was identified in thekidneys, which are its main route of elimination(Fig. 3C). H&E staining and of sectioned pancreas andperitoneal metastases showed the presence of Plec1-expressing tumors in the pancreas and peritoneum(Fig. 3D). These data show that Plec1-targeted imaging

using tPTP functions as a highly specific imaging tool forPDAC, clearly distinguishing PDAC and its metastasesfrom their adjacent normal tissues.

To determine the ability of tPTP to highlight liver metas-tases, AK134 cells were used in a well-knownmodel of livermetastases that form after intrasplenic injection. Animalsinjected with tPTP were imaged 4 hours later, followed bybiodistribution measurements. Metastases in the liver werereadily identified via tPTP-mediated SPECT/CT imaging(Fig. 4A). Biodistribution analysis confirmed the imagingresults, with a 1.7-fold increase in tPTP accumulation inlivers that had metastases over livers from animals devoidof tumors (P < 0.01; Fig. 4B). H&E confirmed the presenceof metastatic disease in livers that were positive via tPTP-mediated SPECT imaging (Fig. 4C). These data show thatPlec1-targeted imaging sing tPTP functions as a highlyspecific imaging tool for PDAC, highlighting PDAC andits metastases.

Discussion

In this study we show that Plec1 expression identifiespreinvasive PanIN III lesions as well as primary and meta-

A B D

C

Figure 3. In vivo imaging of Plec1 in orthotopic PDAC. A, in vitro validation of tPTP. L3.6pl cells were plated on a 96-well plate and incubated with 111In-tPTPand increasing log concentrations of unlabeled tPTP or negative control tetramer. B, mice bearing tumors from orthotopically implanted L3.6pl, AK134 cells,and mice without tumors (null) were injected with 111In-tPTP and imaged via SPECT/CT 4 hours postinjection. Note the accumulation of tPTP in PDAC,allowing the in vivo imaging of tumor in the pancreas and in peritoneal metastases. Coronal (left) and axial (right) SPECT/CT slices through the tumor arepresented. T, tumor; K, kidney; M, peritoneal metastasis. C, after SPECT/CT imaging, animals were sacrificed, organs harvested, and gamma countsassessed. Null data are from both nu/nu and FVB/NJ animals that were injected in the pancreas with saline. D, histology. Animals that had orthotopicallyimplanted tumors or null animals were sacrificed and pancreas and regions of visible peritoneal metastases were removed, embedded, sectioned, and stainedwith H&E (�20 image). PT, primary tumor; PM, peritoneal metastasis.

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static human PDAC. Plec1 expression intensity also clearlydiscriminates cancers from benign conditions, in particularCP. In a preclinical orthotopic mouse model of PDAC,Plec1-targeted noninvasive imaging detects primary andmetastatic PDAC. Taken together, these data suggest thatPlec1 may indeed be an ideal biomarker for PDAC.

The use of Plec1 as a biomarker offers several advan-tages over current clinical diagnostic tools and markers.Unlike CA 19-9, which lacks sensitivity and specificity(7), Plec1 is a specific biomarker for invasive and pre-invasive pancreatic cancer. In contrast to conventionalcross-sectional abdominal imaging (10–13), Plec1-basedimaging would highlight small, currently unidentifiablemetastatic foci preoperatively, thus substantially improv-ing preoperative staging. Due to its ability to potentiallyhighlight preinvasive PDAC, it has the potential to berapidly developed into present-day screening protocolsfor high risk patients with the promise of detecting PDACprior to invasion.

Due to its unique properties, the use of Plec1 as abiomarker for preinvasive and invasive PDAC also holdspromise to be superior to other recently described biomar-kers. Lactose-binding protein, which is overexpressed inacinar cells that surround PDAC, is ideal to detect smallcancers within the pancreas, but cannot detect small metas-tases (24). Neutrophil gelatinase–associated lipocalindelineates PanIN lesions and some invasive cancers, butdoes not identify poorly differentiated adenocarcinoma(23). MUC1 and MUC4 are overexpressed in a subset ofinvasive adenocarcinoma, but their expression does notdistinguish early PanIN I/II from preinvasive (PanIN III)and invasive cancers. MUC4 is also aberrantly expressed in

CP (20, 21). In contrast, Plec1 detects all PDAC and theirsmall metastatic foci as well as the majority of preinvasivecancers (PanIN III) in addition to discriminating PDACfrom CP.

To date, the role of Plec1 overexpression in pancreaticcancer is unknown. Plec1 itself is a cytolinker protein ofthe plakin family. Plakins connect intermediate filamentsto desmosomes and hemidesmosomes, stabilize cellsmechanically, regulate cytoskeleton dynamics, and serveas a scaffolding platform for signaling molecules. Plakinswere first described as essential for skin and skeletalmuscle integrity (26) and mutations of the Plec1 genewere therefore initially identified in skin disease, such asepidermolysis bullosa (27, 28). A recent study, in whichPlec1 was found to interact with the breast cancer sus-ceptibility gene 2 (BRCA2; ref. 29), provides more insightinto the protein’s potential role in cancer. BRCA2 muta-tions are associated with an increased risk of pancreaticcancer (30, 31). BRCA2 itself plays an important role inDNA damage repair and is mainly found in the cellnucleus, but has also been identified in the centrosome.The Plec1/BRCA2 interaction is involved in the regulationof centrosome localization and Plec1 misexpression leadsto displacement of the centrosome. This may contributeto genomic instability and therefore cancer development(29). We found that Plec1 expression is acquired duringthe transition from PanIN II to PanIN III. As lesionsprogress, Plec1 is not retained at the cell membrane,but rather is ubiquitously expressed in the cancer cells.It thus appears that Plec1 overexpression and cytoplasmiclocalization begin at the stage of PanIN III and furtherincrease as lesions progress to invasive cancer.

A

C

B

Figure 4. Plec1 imaging allowsnoninvasive detection of livermetastases. A, AK134 cells, orsaline (null), were injectedintrasplenically to produce livermetastases. Left, mice bearingliver metastasis (LM) from AK134injection. Right, null animalswithout intrasplenic tumor cellinjected. K, kidney. B,biodistribution studies wereperformed on indicated tissuessubsequent to imagingexperiments. *, P < 0.01. C,histology: histologic confirmationof liver metastasis (left); normalliver (right).

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In summary, Plec1 may be the best novel biomarker forPDAC identified to date. Although further studies areneeded to verify Plec1 as a target in humans, strategiesdesigned to image Plec1 could substantially improvedetection and staging, thus contributing to improvedresectability, prognosis, and ultimately survival in pan-creatic cancer.

Disclosure of Potential Conflicts of Interest

The authors declared no potential conflicts of interest.

Acknowledgments

We thank Nabeel Bardeesy for giving us the AK134 cells and thesyngeneic mice. We also thank Sanford Feldman for help with the animals;Sara Adair and Dustin Walters for their help with the orthotopic tumorimplantation; and Nancy Neyhard, Fred Reynolds, and Marc Seaman fortechnical assistance.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 16, 2010; revised September 10, 2010; acceptedOctober 8, 2010; published OnlineFirst November 23, 2010.

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